摘要
目的探究脂肪干细胞(ADSCs)来源的外泌体对2型糖尿病肾病大鼠模型肾脏的作用以及机制。方法以SD大鼠为实验对象,无菌条件下抽提大鼠腹股沟处脂肪组织,进行ADSCs的原代培养,采用油红O染色和流式细胞仪方法对ADSCs进行鉴定。利用超速离心法进行收集外泌体,采用透射电子显微镜和Western blot法对外泌体进行鉴定。构建2型糖尿病肾病大鼠模型,根据实验目的分成3组,即空白对照组,糖尿病肾病组和外泌体治疗组。处理12周后无痛苦处死大鼠,无菌条件下摘取肾脏,利用HE染色和透射电镜法观察肾脏的形态,利用RT-PCR和Western blot法检测肾脏组织中凋亡相关蛋白Bcl-2,Bad和Bax的表达。结果原代ADSCs细胞呈多角型,贴壁生长,油红O染色阳性,ADSCs细胞表面蛋白CD90和CD105的表达分别为86.7%和91.4%;CD31和CD45的表达分别为0.4%和1.2%。外泌体为直径50~150nm的双层膜囊泡状结构,外泌体的分子标志物CD81和CD63表达阳性。外泌体治疗组大鼠肾脏肾小球较为规则,系膜未见明显增生。大鼠肾脏组织抗凋亡因子Bcl-2的相对表达量对照组明显高于糖尿病肾病组,外泌体治疗组明显高于糖尿病肾病组。大鼠肾脏组织凋亡相关因子Bad和Bax的相对表达量对照组明显低于糖尿病肾病组,外泌体治疗组明显低于糖尿病肾病组。结论ADSCs来源的外泌体通过抑制细胞凋亡和系膜组织增生起到保护肾功能的作用。
Objective To investigate the effect and mechanism of adipose-derived stem cells(ADSCs) derived exosomes on kidney of type 2 diabetic nephropathy rats. Methods Adipose tissue was extracted from the inguinal region of SD rats under sterile condition, and primary culture of ADSCs was carried out. ADSCs were identified by oil red O staining and flow cytometry. The exosomes were collected by ultracentrifugation and identified by transmission electron microscopy and Western blot. The rat model of type 2 diabetic nephropathy was established and divided into three groups according to the experimental purpose, namely blank control group, diabetic nephropathy group and exosome treatment group. After 12 weeks of treatment, rats were sacrificed painlessly. The kidneys were harvested under aseptic conditions. The morphology of kidneys was observed by HE staining and transmission electron microscope. The expression of apoptosis-related proteins Bcl-2, Bad and Bax was detected by RT-PCR and Western blot. Results The primary ADSCs cells were polygonal, adherent and oil red O positive, the expressions of CD90 and CD105 were 86.7% and 91.4% respectively, and 0.4% and 1.2% for CD31 and CD45 respectively. The exosome is a bilayer membrane vesicle with a diameter of 50~150 nm. The molecular markers CD81 and CD63 for exosomes were positive. The glomerulus of the kidney was irregular in diabetic nephropathy group, but regular in exosome group. The relative expression of anti-apoptotic factor Bcl-2 was significantly higher in the control group than in diabetic nephropathy group, and significantly higher in exosome treatment group than in the diabetic nephropathy group. The relative expression of Bad and Bax in kidney tissue was significantly lower in control group than in diabetic nephropathy group, and lower in exosome group than in diabetic nephropathy group. Conclusion The recovered effect of ADSCs-derived exosomes on renal function is related to inhibiting apoptosis and mesangial tissue proliferation in diabetic nephropathy SD rats.
作者
陈晶
刘清岩
张海宁
王颜刚
CHEN Jing;LIU Qing-yan;ZHANG Hai-ning;WANG Yan-gang(Department of Endocrinology,Affiliated Hospital of Qingdao University,Qingdao 266003,China)
出处
《解剖科学进展》
2020年第4期369-373,共5页
Progress of Anatomical Sciences
基金
国家自然科学基金(81672197)。
关键词
脂肪干细胞
外泌体
糖尿病肾病
透射电镜
SD大鼠
adipose-derived stem cells
exosomes
diabetic nephropathy
transmission electron microscope
SD rat
